Edible Matrix Code with Photogenic Silk Proteins

Counterfeit medicines are a healthcare security problem, posing not only a direct threat to patient safety and public health but also causing heavy economic losses. Current anticounterfeiting methods are limited due to the toxicity of the constituent materials and the focus of secondary packaging level protections. We introduce an edible, imperceptible, and scalable matrix code of information representation and data storage for pharmaceutical products. This matrix code is digestible as it is composed of silk fibroin genetically encoded with fluorescent proteins produced by ecofriendly, sustainable silkworm farming. Three distinct fluorescence emission colors are incorporated into a multidimensional parameter space with a variable encoding capacity in a format of matrix arrays. This code is smartphone-readable to extract a digitized security key augmented by a deep neural network for overcoming fabrication imperfections and a cryptographic hash function for enhanced security. The biocompatibility, photostability, thermal stability, long-term reliability, and low bit error ratio of the code support the immediate feasibility for dosage-level anticounterfeit measures and authentication features. The edible code affixed to each medicine can serve as serialization, track and trace, and authentication at the dosage level, empowering every patient to play a role in combating illicit pharmaceuticals.

Screening and production of transgenic silkworms. We immersed eggs obtained from female moths in a HCl solution with a specific gravity of 1.0955 at 25 °C for 30 minutes, followed by rinsing with deionized water and drying. The transition vectors p3×P3-DsRed2-pFibH-eCFP, p3×P3-DsRed2-pFibH-eGFP, or p3×P3-eGFP-pFibH-mKate2 and the helper vector pHA3PIG were dissolved in an aqueous solution of 5 mM KCl and 0.5 mM PBS (pH 7.2) at a concentration of 0.2 g L -1 and mixed at a ratio of 1:1, respectively. The mixture solution of 5 − 10 nL was injected into pre-blastoderm embryos at 2 -8 hours post-oviposition using an IM300 microinjector (Narishige Scientific Instrument Lab., Tokyo, Japan). DNA-injected embryos were allowed to develop at 25 °C in moist chambers until hatching. The hatched larvae (Generation 0; G0) were reared and permitted to mate with each other. The resulting embryos (G1) and larvae were screened under a fluorescence microscopy (Leica, Wetzlar, Germany) using a red filter for eCFP and eGFP and a green filter for mKate2. The G1 hatched larvae (i.e., silkworms) were reared in groups and were fed with fresh mulberry leaves under the condition of 24 − 27 °C and 70 − 90% relative humidity.
Silk cocoon degumming. We eliminated sericin from silk cocoons, following the commonly available protocol of minimizing the heat-induced denaturation of fluorescent proteins (i.e., eCFP, eGFP, and mKate2) in silk. [1][2][3] The silk cocoons were treated several times with a 0.2% NaHCO3 solution at a temperature less than 50 °C under an induced low pressure 620 mmHg, subsequently washed with deionized water several times. The degummed cocoons were naturally dried in the dark under ambient conditions. Scanning electron microscopy (SEM). Structural morphologies were characterized by a SEM system (FEI Quanta 3D FEG) at 5 kV.
Patterning of micrograting arrays on non-fluorescent white silk fibroin films via soft imprint lithography. To fabricate a white silk fibroin film patterned with micrograting arrays as a thin substrate of edible matrix codes, we used a 2-inch sapphire substrate patterned with a conical micrograting structure consisting of two-dimensional (2D) periodic hexagonal arrays. The micrograting structure with an average height of 1.5 µm and an average period of 2.9 µm has strong light diffraction, while maintaining a high transmittance value over a wide wavelength range of 300 − 1100 nm. 4,5 As an imprint stamp, an elastomeric polydimethylsiloxane (PDMS) template was utilized because of its excellent formability for micropatterns. Sylgard 184, which is composed of a silicone "T-resin" crosslinked by a mixture of vinyl-terminated PDMS and trimethylsiloxyterminated poly(methylhydro-siloxane) polymers with a ratio of 10:1 (base:agent), was poured on the patterned sapphire substrate and was cured at 75 °C for 2 hours. PDMS stamps were carefully separated from the patterned substrate, creating inverse conical micrograting patterned structures. The 5% (w v −1 ) white silk fibroin-dissolved solution was poured on the PDMS stamp, and was cured under ambient conditions. The cured silk fibroin films were cautiously peeled off from PDMS stamps, producing a silk fibroin film patterned with conical micrograting arrays. The height and diameter of gratings are 1.4 and 2.7 µm with a distance between adjacent gratings of 2.9 µm. The thickness of patterned silk fibroin films was 100 ± 5 µm.
Construction of a custom-built mobile application (app) for reading an edible code. To read an edible code using a smartphone, we built a customized mobile app designed with Android in Java (Android studio 4.0 integrated development environment version 2.1.2, Android SDKsoftware development kit, and Android API Level 23), using the Open Source Computer Vision (OpenCV) library. The mobile app allows device control, image capture, preview, authentication, and hyperlink (product information).
Numerical modeling of optical diffraction from a silk firoin film patterned with micrograting arrays. We simulated optical diffraction of a silk fibroin film patterned with micrograting arrays using the finite-difference time-domain (FDTD) method (FullWAVE, Rsoft Design Group, Ossining, NY, USA) ( Figure S4c). The conical microgratings on the silk fibroin film were represented by a periodic geometry in the Cartesian coordinate system by a scalarvalued function of two variables f(x, z) for simplicity. 4 Assuming that the incident light enters from air into the patterned silk fibroin film at normal incidence, the amplitude of y-polarized electric field (Ey) was calculated for the incident plane wave with a Gaussian beam profile, which was normalized at λ = 532 nm. The height and period of gratings were kept at 1.4 μm and 2.9 μm, respectively. The thickness of the silk fibroin film was set to be 100 μm. The refractive index of silk fibroin was assumed to be 1.56, and the extinction coefficient was not considered because it is negligible. 6 S4 Photostability of fluorescent silk fibroin films under alcohol treatments. We examined the effect of alcohol solvent on the photostability of fluorescent silk films using eGFP silk fibroin films with a thickness of 70 µm and a size of 9 × 9 mm 2 ( Figure S6). 200 proof ethyl alcohol (ethanol) was used as a test solvent. The eGFP silk fibroin films were soaked in the prepared ethanol solvents with different concentrations of 10 -99%. For comparison, deionized water (0% ethanol) was also utilized. The photostability and deformation of eGFP silk fibroin films immersed in a solvent were monitored by taking fluorescence images at a center wavelength of 525 nm under an excitation wavelength of 470 nm.
Reading of edible codes using a custom-built imaging system and a smartphone. We acquired fluorescence images of edible codes using a customized imaging system and a smartphone (model: Samsung Galaxy S21) with a custom-built mobile app. The imaging area of the imaging system is ~ 15 × 15 mm 2 . For optical excitation sources of the imaging system, we used ultraviolet, blue, and green light-emitting diodes (LEDs) purchased from Thorlabs Inc. , and 20 seconds for eCFP silk, eGFP silk, and mKate2 silk, respectively. The optical power was kept at 3, 5, and 8 μW mm −2 for 415-nm, 470-nm, and 530nm LEDs at the surface of the samples, respectively. To read edible codes using a custom-built app, a smartphone was equipped with excitation (ex = 470 nm) and emission (em = 525 nm) optical filters ( Figure S11).

Photostability of fluorescent silk fibroin films under simulated daylight illumination.
We tested the photostability of fluorescent silk fibroin films composing edible codes using a CIE Standard Illuminant LED light source with a color temperature of 6500 K (also known as D65) that mimics daylight illumination. Fluorescent silk fibroin films regenerated from eCFP silk, eGFP silk, and mKate2 silk had a thickness of 70 µm and a size of 9 × 9 mm 2 . To conduct accelerated photobleaching over a short period of time, fluorescent silk fibroin films were irradiated by a high intensity of 5000 lx, which is 10 times stronger than the recommended light level intensity of 500 lx at a typical office workspace. 7 The optical intensity of white LEDs was measured using a commercial light meter (LX1330B-V, Dr. Meter). The fluorescence emission of each fluorescent silk fibroin film was monitored at each peak emission wavelength of 485 nm, 525 nm, and 625 nm under the corresponding excitation wavelength of 415 nm, 470 nm, and 530 nm for eCFP silk, eGFP silk, and mKate2 silk, respectively. Reduction in the fluorescence intensity due to photobleaching was quantified by the fluorescence intensity normalized by the initial value before illumination ( Figure S13).

Key extraction performance of edible codes under thermal treatments.
We explored the thermal stability of fluorescent silk fibroin films and edible codes. Fluorescent silk fibroin films and edible codes were placed in an oven at different temperatures of 30 − 90 °C for three hours ( Figure S14). The eCFP silk, eGFP silk, and mKate2 silk fibroin films with a thickness of 70 µm and a diameter of 13 mm were tested. The fluorescence emission of each fluorescent silk fibroin film was monitored at each peak emission wavelength of λem = 485 nm, 525 nm, and 625 nm under excitation of λex = 415 nm, 470 nm, and 530 nm for eCFP silk, eGFP silk, and mKate2 silk, respectively. Reduction in the fluorescence intensity due to heat-induced denaturation was quantified by the fluorescence intensity normalized by the initial value at room temperature (i.e., 23 °C). For edible codes with a 7 × 7 matrix array, we calculated bit error ratios of output keys extracted from edible codes relative to the corresponding fluorescent code patterns. ReLU: rectified linear unit    Numerical experiments of the calculated electric field (Ey) for the incident light propagating from air to a bare silk fibroin film (left) and a micrograting patterned silk fibroin film (right) under the light illumination at a wavelength (λ) of 532 nm. This simulation is conducted using the FDTD method. The electric field distribution through the conical micrograting arrays supports the experimental result that light diffraction masks the embedded code pattern, enhancing the covertness of edible matrix codes (Fig. 2h). Figure S5. Photograph of representative edible codes with 5 × 5, 7 × 7, and 9 × 9 matrix arrays. The size of an edible matrix code can be varied by controlling the number of codes, resulting in an encoding capacity of 2 75 (≈ 3.77 × 10 22 ), 2 147 (≈ 1.78 × 10 44 ), and 2 243 (≈ 1.41 × 10 73 ) for 5 × 5, 7 × 7, and 9 × 9 matrix codes, respectively. In these cases, the digitized key size extracted from 5 × 5, 7 × 7, and 9 × 9 matrix codes with three fluorescence colors are 75 (= 5 × 5 × 3), 147 (= 7 × 7 × 3), and 243 (9 × 9 × 3), respectively. With the individual square code pattern size of 700 × 700 µm 2 , the corresponding sizes of 5 × 5, 7 × 7, and 9 × 9 matrix codes are 7 × 7, 9 × 9, and 11 × 11 mm 2 , respectively.        The fluorescence intensity is normalized by the value before illumination (0 hour) at each maximum emission peak (i.e., 485 nm for eCFP silk, 525 nm for eGFP silk, and 625 nm for mKate2 silk). If a currently available pharmaceutical (dark or opaque) packaging with light protection is used, the shelf life will be significantly extended. (b) Illumination spectrum of a CIE Standard Illuminant LED light source with a color temperature of 6500 K (also known as D65) used in this accelerated photobleaching experiment and absorption spectra of eCFP silk, eGFP silk, and mKate2 silk fibroin films. The D65 LED light source covers the absorption wavelength range of all fluorescent silk fibroin films and has a high illumination intensity of 5000 lx, which is 10 times higher than the recommended office workspace light intensity (i.e., 500 lx).  and extracted output keys (right) acquired after 360 days, compared with those in Fig. 3a. The same optical set of excitation and emission is used: λex = 415 nm and λem = 460 nm for eCFP silk (cyan); 470 nm and 525 nm for eGFP silk (green); and 530 nm and 630 nm for mKate2 silk (red). The storage condition is the ambient dark environment (i.e., 23 ± 2 °C and 30 -40% relative humidity).